Altered promoter selection by a novel form of Bacillus subtilis RNA polymerase

AUTOR(ES)

Jaehning, Judith A.

RESUMO

Bacillus subtilis RNA polymerase holoenzyme prepared by several standard methods utilizes bacteriophage T7 ΔD111 DNA as an efficient template. The major RNA products are specific transcripts from T7 promoters A1 and C; these promoters are also efficiently utilized by RNA polymerases purified from a wide range of other bacterial species [Wiggs, J., Bush, J. & Chamberlin, M. (1979) Cell 16, 97-109]. In contrast, B. subtilis RNA polymerase preparations purified by a modification of the method of Burgess and Jendrisak (designated fraction 5) utilize T7 ΔD111 promoters A1 and C and an additional promoter site, J, which has been located at 90.6% on the standard T7 physical map. This promoter is not used by B. subtilis core RNA polymerase or by RNA polymerase from any other bacterial species we have tested. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of fraction 5 RNA polymerase shows that it contains B. subtilis components σ and δ and a polypeptide of Mr 92,000 in addition to the B. subtilis β, β′, and α subunits. Chromatography of fraction 5 on single-stranded DNA-cellulose gives an enzyme fraction, Bs I, that is indistinguishable from B. subtilis RNA polymerase holoenzyme both in its peptide composition (ββ′α2σ) and in the selective transcription of only T7 RNAs A1 and C. Chromatography of fraction 5 on phosphocellulose yields an enzyme fraction, Bs II, devoid of σ subunit but containing the Mr 92,000 peptide and traces of δ. This fraction synthesizes predominantly T7 J RNA in vitro together with traces of T7 A1 and C RNAs. Hence, B. subtilis RNA polymerase fraction Bs II appears to contain a form of RNA polymerase that can transcribe selectively without detectable amounts of B. subtilis σ subunit and that utilizes a promoter site not used by other known bacterial RNA polymerases. The structural basis for this specificity is not yet known.

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